Distinct functional properties of the TAP subunits coordinate the nucleotide-dependent transport cycle

BACKGROUND: The transporter associated with antigen processing (TAP) consists of two polypeptides, TAP1 and TAP2. TAP delivers peptides into the ER and forms a "loading complex" with MHC class I molecules and accessory proteins. Our previous experiments indicated that nucleotide binding to TAP plays a critical role in the uptake of peptide and the release of assembled class I molecules. To investigate whether the conserved nucleotide binding domains (NBDs) of TAP1 and TAP2 are functionally equivalent, we created TAP variants in which only one of the two ATP binding sites was mutated. RESULTS: Mutations in the NBDs had no apparent effect on the formation of the loading complex. However, both NBDs had to be functional for peptide uptake and transport. TAP1 binds ATP much more efficiently than does TAP2, while the binding of ADP by the two chains is essentially equivalent. Peptide-mediated release of MHC class I molecules from TAP was blocked only when the NBD of TAP1 was disrupted. A different NBD mutation that does not affect nucleotide binding has strikingly different effects on peptide transport activity depending on whether it is present in TAP1 or TAP2. CONCLUSIONS: Our findings indicate that ATP binding to TAP1 is the initial step in energizing the transport process and support the view that ATP hydrolysis at one TAP chain induces ATP binding at the other chain; this leads to an alternating and interdependent catalysis of both NBDs. Furthermore, our data suggest that the peptide-mediated undocking of MHC class I is linked to the transport cycle of TAP by conformational signals arising predominantly from TAP1.     

Identification of domain boundaries within the N-termini of TAP1 and TAP2 and their importance in tapasin binding and tapasin-mediated increase in peptide loading of MHC class I

Before exit from the endoplasmic reticulum (ER), MHC class I molecules transiently associate with the transporter associated with antigen processing (TAP1/TAP2) in an interaction that is bridged by tapasin. TAP1 and TAP2 belong to the ATP-binding cassette (ABC) transporter family, and are necessary and sufficient for peptide translocation across the ER membrane during loading of MHC class I molecules. Most ABC transporters comprise a transmembrane region with six membrane-spanning helices. TAP1 and TAP2, however, contain additional N-terminal sequences whose functions may be linked to interactions with tapasin and MHC class I molecules. Upon expression and purification of human TAP1/TAP2 complexes from insect cells, proteolytic fragments were identified that result from cleavage at residues 131 and 88 of TAP1 and TAP2, respectively. N-Terminally truncated TAP variants lacking these segments retained the ability to bind peptide and nucleotide substrates at a level comparable to that of wild-type TAP. The truncated constructs were also capable of peptide translocation in vitro, although with reduced efficiency. In an insect cell-based assay that reconstituted the class I loading pathway, the truncated TAP variants promoted HLA-B*2705 processing to similar levels as wild-type TAP. However, correlating with the observed reduction in tapasin binding, the tapasin-mediated increase in processing of HLA-B*2705 and HLA-B*4402 was lower for the truncated TAP constructs relative to the wild type. Together, these studies indicate that N-terminal domains of TAP1 and TAP2 are important for tapasin binding and for optimal peptide loading onto MHC class I molecules.     

The human cytomegalovirus gene product US6 inhibits ATP binding by TAP

Human cytomegalovirus (HCMV) encodes several genes that disrupt the major histocompatibility complex (MHC) class I antigen presentation pathway. We recently described the HCMV-encoded US6 gene product, a 23 kDa endoplasmic reticulum (ER)-resident type I integral membrane protein that binds to the transporter associated with antigen processing (TAP), inhibits peptide translocation and prevents MHC class I assembly. The functional consequence of this inhibition is to prevent the cell surface expression of class I bound viral peptides and their recognition by HCMV-specific cytotoxic T cells. Here we describe a novel mechanism of action for US6. We demonstrate that US6 inhibits the binding of ATP by TAP1. This is a conformational effect, as the ER lumenal domain of US6 is sufficient to inhibit ATP binding by the cytosolic nucleotide binding domain of TAP1. US6 also stabilizes TAP at 37 degrees C and prevents conformational rearrangements induced by peptide binding. Our findings suggest that the association of US6 with TAP stabilizes a conformation in TAP1 that prevents ATP binding and subsequent peptide translocation.     

Antigen presentation in Syrian hamster cells: substrate selectivity of TAP controlled by polymorphic residues in TAP1 and differential requirements for loading of H2 class I molecules

Expression of mouse major histocompatibility complex (MHC) class I molecules in different cell lines derived from Syrian hamsters has revealed antigen presentation deficiencies of some H2 allelic products in two cell lines (BHK and NIL-2) which were overcome by transient expression of the rat transporter associated with antigen processing (TAP; Lobigs et al. 1995). Here we show that in both cell lines the endogenous MHC class I cell surface expression was completely down-regulated. Lymphokine treatment induced endogenous and recombinant mouse MHC class I cell surface expression to levels similar to that in other Syrian hamster cell lines competent for antigen presentation through transduced H2 molecules. Accordingly, constitutive downregulation of expression of accessory molecules of the MHC class I pathway can reveal differences between H2 class I alleles in antigen presentation not encountered when the expression levels are augmented. In addition to the differential expression of MHC class I pathway genes, two cell lines representing competent (FF) and defective (BHK) antigen presentation phenotypes for mouse class I MHC restriction elements demonstrated substantial sequence polymorphism in Tap1 but not Tap2. Cytokine-treated FF or BHK cells and human TAP-deficient T2 cells transfected with FF or BHK TAP1 in combination with FF TAP2 differed in their preference for C-terminal peptide residues, as shown by an in vitro peptide transport assay. Thus, polymorphic residues in TAP1 can influence the substrate selectivity of the Syrian hamster peptide transporter.     

Functional cysteine-less subunits of the transporter associated with antigen processing (TAP1 and TAP2) by de novo gene assembly

Within the adaptive immune system the transporter associated with antigen processing (TAP) plays a pivotal role in loading of peptides onto major histocompatibility (MHC) class I molecules. As a central tool to investigate the structure and function of the TAP complex, we created cysteine-less human TAP subunits by de novo gene synthesis, replacing all 19 cysteines in TAP1 and TAP2. After expression in TAP-deficient human fibroblasts, cysteine-less TAP1 and TAP2 are functional with respect to adenosine triphosphate (ATP)-dependent peptide transport and inhibition by ICP47 from herpes simplex virus. Cysteine-less TAP1 and TAP2 restore maturation and intracellular trafficking of MHC class I molecules to the cell surface.     

TAP1 and TAP2 gene polymorphisms in childhood cystic echinococcosis

The incidence of cystic echinococcosis (CE) due to Echinococcus granulosus is as high as 2000–2500 patients per year in Turkey. Whether genetic characteristics of the Turkish population cause a tendency to the disease is currently unknown. We aimed at studying the role of TAP gene polymorphisms in Turkish children with cystic echinococcosis. For an overview of allelic distribution of TAP1 and TAP2 genes, genotypes of 85 patients with CE and 100 controls were studied. To determine the genotype–phenotype correlation, 81 of the patients whose clinical data were available were analyzed. For TAP1-637, Asp/Gly heterozygosity was significantly more prevalent in CE patients than in controls (20 vs. 4%, odds ratio 6.0), while Gly/Gly homozygosity was less frequent (5 vs. 14%). For TAP2-379, Ile/Val heterozygosity was significantly more prevalent in CE patients than in controls (14 vs. 1%, odds ratio 16.27), while Ile/Ile homozygosity was less frequent (13 vs. 25%). TAP1-637 and TAP2-379 polymorphisms may have a role in causing genetic tendency for CE in children. The data may reflect the genetic properties of the Turkish population or may reveal the minor role of TAP gene polymorphisms in CE.     

Powering the peptide pump: TAP crosstalk with energetic nucleotides

ATP-binding cassette (ABC) transporters represent a large family of membrane-spanning proteins that have a shared structural organization and conserved nucleotide-binding domains (NBDs). They transport a large variety of solutes, and defects in these transporters are an important cause of human disease. TAP (tmacr;ransporter associated with āntigen pmacr;rocessing) is a heterodimeric ABC transporter that uses nucleotides to drive peptide transport from the cytoplasm into the endoplasmic reticulum lumen, where the peptides then bind major histocompatibility complex (MHC) class I molecules. TAP plays an essential role in the MHC class I antigen presentation pathway. Recent studies show that the two NBDs of TAP fulfil distinct functions in the catalytic cycle of this transporter. In this opinion article, a model of alternating ATP binding and hydrolysis is proposed, in which nucleotide interaction with TAP2 primarily controls substrate binding and release, whereas interaction with TAP1 controls structural rearrangements of the transmembrane pathway. Viral proteins that inhibit TAP function cause arrests at distinct points of this catalytic cycle.     

Polymorphism of the TAP1 gene in Polish patients with psoriasis vulgaris

Psoriasis vulgaris is a HLA-associated common and persistent inflammatory skin disease of unknown aetiology. The transporters associated with antigen processing (TAP) genes are polymorphic genes located in the HLA class II region and due to their essential involvement in class I antigen presentation might be additional susceptibility genes to psoriasis. To investigate the possible involvement of the TAP1 gene in the pathogenesis of psoriasis, we analysed its polymorphism in 169 Polish patients with psoriasis vulgaris and compared them with 66 healthy controls. The frequency of TAP1*D was significantly increased in the patients, compared to the control group. The TAP alleles were also analysed with respect to the age of onset of psoriasis in the patients but no significant differences were recorded. In conclusion, our data suggest that the TAP1*D allele could lead to genetic susceptibility to psoriasis vulgaris in Poles.     

TAP1 and TAP2 polymorphism in coeliac disease

Coeliac disease is strongly associated with HLA-DQ2, but it is possible that additional major histocompatibility complex genes also confer disease susceptibility. Encoded close to HLA-DQ are two genes, TAP1 and TAP2, whose products are believed to transport antigenic peptides from the cytoplasm into the endoplasmic reticulum. Comparison of 81 coeliac disease patients with caucasoid controls revealed an increased frequency of the alleles TAP1A and TAP2A in the patient population. However, no significant difference was found when patients were compared with HLA-DR and -DQ matched controls, indicating linkage disequilibrium between TAP1A, TAP2A, and HLA-DQ2. The TAP gene products do not have a major influence on susceptibility or resistance to coeliac disease in a Northern European Caucasoid population. Correspondence to: S. H. Powis.     

Competition for access to the rat major histocompatibility complex class I peptide-loading complex reveals optimization of peptide cargo in the absence of transporter associated with antigen processing (TAP) association

Major histocompatibility complex (MHC) class I molecules load peptides in the endoplasmic reticulum in a process during which the peptide cargo is normally optimized in favor of stable MHC-peptide interactions. A dynamic multimolecular assembly termed the peptide-loading complex (PLC) participates in this process and is composed of MHC class I molecules, calreticulin, ERp57, and tapasin bound to the transporter associated with antigen processing (TAP) peptide transporter. We have exploited the observation that the rat MHC class I allele RT1-Aa, when expressed in the rat C58 thymoma cell line, effectively competes and prevents the endogenous RT1-Au molecule from associating with TAP. However, stable RT1-Au molecules are assembled efficiently in competition with RT1-Aa, demonstrating that cargo optimization can occur in the absence of TAP association. Defined mutants of RT1-Aa, which do not allow formation of the PLC, fail to become thermostable in C58 cells. Wild-type RT1-Aa, which does allow PLC formation, also fails to become thermostable in this cell line, which carries the rat TAPB transporter that supplies peptides incompatible for RT1-Aa binding. Full optimization of RT1-Aa requires the presence of the TAP2A allele, which is capable of supplying suitable peptides. Thus, formation of the PLC alone is not sufficient for optimization of the MHC class I peptide cargo.     

Alleles and haplotypes of the MHC-encoded ABC transporters TAP1 and TAP2

TAP1 and TAP2 are two major histocompatibility complex (MHC) genes, located between HLA-DP and -DQ, whose products form a transporter molecule involved in endogenous antigen processing. Polymorphic residues have been described in both genes and, in this study, we have identified another polymorphic site within the adenosine triphosphate (ATP)-binding domain of TAP2. We have used the amplification refractoru mutation system (ARMS) polymerase chain reaction (PCR) to characterize TAP1 and TAP2 alleles and haplotypes in a reference panel of 115 homozygous typing cell lines. Of four possible TAP1 alleles, we observed three, and of eight possible TAP2 alleles, we observed five. Among 88 (homozygous typing cells) (HTCs) homozygous at HLA-DR, -DQ and TP, 80 were also homozygous at TAP1 and TAP2. Of 27 HTCs homozygous at HLA-DR and -DQ, but heterozygous at -DP, 14 were homozygous at TAP1 or TAP2 and 13 heterozygous, consistent with recombination taking place either side of the TAP loci. Of the fifteen possible combinations of TAP1 and TAP2 alleles, we observed eleven, each at a frequency similar to that predicted by individual allele frequencies. In this ethnically heterogeneous panel there is no indication that particular combinations of TAP1 and TAP2 have been maintained together. Correspondence to: S. H. Powis.     

Impaired transporter associated with antigen processing (TAP) function attributable to a single amino acid alteration in the peptide TAP subunit TAP1

The heterodimeric peptide transporter TAP belongs to the ABC transporter family. Sequence comparisons with the P-glycoprotein and cystic fibrosis transmembrane conductance regulator and the functional properties of selective amino acids in these ABC transporters postulated that the glutamic acid at position 263 and the phenylalanine at position 265 of the TAP1 subunit could affect peptide transporter function. To define the role of both amino acids, TAP1 mutants containing a deletion or a substitution to alanine at position 263 or 265 were generated and stably expressed in murine and human TAP1(-/-) cells. The different TAP1 mutants were characterized in terms of expression and function of TAP, MHC class I surface expression, immune recognition, and species-specific differences. The phenotype of murine and human cells expressing human TAP1 mutants with a deletion or substitution of Glu(263) was comparable to that of TAP1(-/-) cells. In contrast, murine and human TAP1 mutant cells containing a deletion or mutation of Phe(265) of the TAP1 subunit exhibit wild-type TAP function. This was associated with high levels of MHC class I surface expression and recognition by specific CTL, which was comparable to that of wild-type TAP1-transfected control cells. Thus, biochemical and functional evidence is presented that the Glu(263) of the TAP1 protein, but not the Phe(265), is critical for proper TAP function.     

Modulation of the antigenic peptide transporter TAP by recombinant antibodies binding to the last five residues of TAP1

The transporter associated with antigen processing (TAP) plays a pivotal role in the major histocompatibility complex (MHC) class I mediated immune response against infected or malignantly transformed cells. It belongs to the ATP-binding cassette (ABC) superfamily and consists of TAP1 (ABCB2) and TAP2 (ABCB3), each of which possesses a transmembrane and a nucleotide-binding domain (NBD). Here we describe the generation of recombinant Fv and Fab antibody fragments to human TAP from a hybridoma cell line expressing the TAP1-specific monoclonal antibody mAb148.3. The epitope of the antibody was mapped to the very last five C-terminal amino acid residues of TAP1 on solid-supported peptide arrays. The recombinant antibody fragments were heterologously expressed in Escherichia coli and purified to homogeneity from periplasmic extracts by affinity chromatography. The monoclonal and recombinant antibodies bind with nanomolar affinity to the last five C-terminal amino acid residues of TAP1 as demonstrated by ELISA and surface plasmon resonance. Strikingly, the recombinant antibody fragments confer thermal stability to the heterodimeric TAP complex. At the same time TAP is arrested in a peptide transport incompetent conformation, although ATP and peptide binding to TAP are not affected. Based on our results we suggest that the C terminus of TAP1 modulates TAP function presumably as part of the dimer interface of the NBDs.     

Quantifying recruitment of cytosolic peptides for HLA class I presentation: impact of TAP transport

MHC class I ligands are recruited from the cytosolic peptide pool, whose size is likely to depend on the balance between peptide generation by the proteasome and peptide degradation by downstream peptidases. We asked what fraction of this pool is available for presentation, and how the size of this fraction is modulated by peptide affinity for the TAP transporters. A model epitope restricted by HLA-A2 and a series of epitope precursors with N-terminal extensions by single residues modifying TAP affinity were expressed in a system that allowed us to monitor and modulate cytosolic peptide copy numbers. We show that presentation varies strongly according to TAP affinities of the epitope precursors. The fraction of cytosolic peptides recruited for MHC presentation does not exceed 1% and is more than two logs lower for peptides with very low TAP affinities. Therefore, TAP affinity has a substantial impact on MHC class I Ag presentation.     

The varicellovirus UL49.5 protein blocks the transporter associated with antigen processing (TAP) by inhibiting essential conformational transitions in the 6+6 transmembrane TAP core complex

TAP translocates virus-derived peptides from the cytosol into the endoplasmic reticulum, where the peptides are loaded onto MHC class I molecules. This process is crucial for the detection of virus-infected cells by CTL that recognize the MHC class I-peptide complexes at the cell surface. The varicellovirus bovine herpesvirus 1 encodes a protein, UL49.5, that acts as a potent inhibitor of TAP. UL49.5 acts in two ways, as follows: 1) by blocking conformational changes of TAP required for the translocation of peptides into the endoplasmic reticulum, and 2) by targeting TAP1 and TAP2 for proteasomal degradation. At present, it is unknown whether UL49.5 interacts with TAP1, TAP2, or both. The contribution of other members of the peptide-loading complex has not been established. Using TAP-deficient cells reconstituted with wild-type and recombinant forms of TAP1 and TAP2, TAP was defined as the prime target of UL49.5 within the peptide-loading complex. The presence of TAP1 and TAP2 was required for efficient interaction with UL49.5. Using deletion mutants of TAP1 and TAP2, the 6+6 transmembrane core complex of TAP was shown to be sufficient for UL49.5 to interact with TAP and block its function. However, UL49.5-induced inhibition of peptide transport was most efficient in cells expressing full-length TAP1 and TAP2. Inhibition of TAP by UL49.5 appeared to be independent of the presence of other peptide-loading complex components, including tapasin. These results demonstrate that UL49.5 acts directly on the 6+6 transmembrane TAP core complex of TAP by blocking essential conformational transitions required for peptide transport.     

Tapasin interacts with the membrane-spanning domains of both TAP subunits and enhances the structural stability of TAP1 x TAP2 Complexes

The transporter associated with antigen processing (TAP) proteins are involved in transport of peptides from the cytosol into the endoplasmic reticulum. Two subunits, TAP1 and TAP2, are necessary and sufficient for peptide binding and peptide translocation across the endoplasmic reticulum membrane. TAP1 and TAP2 contain an N-terminal hydrophobic membrane-spanning region and a C-terminal nucleotide binding domain. Tapasin is an endoplasmic reticulum resident protein that has been found associated with the TAP subunits and shown to increase expression levels of TAP. Here we investigated TAP-tapasin interactions and their effects on TAP function in insect cells. We show tapasin binding to both TAP1 and TAP2 and to the corresponding nucleotide binding domain-exchanged chimeras as well as to a truncated TAP1.TAP2 complex containing just the membrane-spanning regions of TAP1 and TAP2. However, tapasin interactions with either the truncated TAP construct containing just the nucleotide binding domain are not observed. Tapasin is not required for high affinity peptide binding to TAP1.TAP2 complexes, and in fact, the presence of tapasin slightly reduces the affinity of TAP complexes for peptides. However, at near physiological temperatures, both tapasin and nucleotides stabilize the peptide binding site of TAP1.TAP2 complexes against inactivation, and enhanced thermostability of both TAP subunits is observed in the presence of tapasin. The enhanced structural stability of TAP1.TAP2 complexes in the presence of tapasin might explain the observations that tapasin increases TAP protein expression levels in mammalian cells.     

Catalytic site modifications of TAP1 and TAP2 and their functional consequences

The transporter associated with antigen processing (TAP), a member of the ATP binding cassette (ABC) family of transmembrane transporters, transports peptides across the endoplasmic reticulum membrane for assembly of major histocompatibility complex class I molecules. Two subunits, TAP1 and TAP2, are required for peptide transport, and ATP hydrolysis by TAP1.TAP2 complexes is important for transport activity. Two nucleotide binding sites are present in TAP1.TAP2 complexes. Compared with other ABC transporters, the first nucleotide binding site contains non-consensus catalytic site residues, including Asp(668) in the Walker B region of TAP1 (in place of a highly conserved glutamic acid), and Gln(701) in the switch region of TAP1 (in place of a highly conserved histidine). At the second nucleotide binding site, a glutamic acid (TAP2 Glu(632)) follows the Walker B motif, and the switch region contains a histidine (TAP2 His(661)). We found that alterations at Glu(632) and His(661) of TAP2 significantly reduced peptide translocation and/or TAP-induced major histocompatibility complex class I surface expression. Alterations of TAP1 Asp(668) alone or in combination with TAP1 Gln(701) had only small effects on TAP activity. Thus, the naturally occurring Asp(668) and Gln(701) alterations of TAP1 are likely to contribute to attenuated catalytic activity at the first nucleotide binding site (the TAP1 site) of TAP complexes. Due to its enhanced catalytic activity, the second nucleotide binding site (the TAP2 site) appears to be the main site driving peptide transport. A mechanistic model involving one main active site is likely to apply to other ABC transporters that have an asymmetric distribution of catalytic site residues within the two nucleotide binding sites.     

Regulation of MHC class I heterodimer stability and interaction with TAP by tapasin

 Major histocompatibility complex (MHC) class I molecules are heterodimers of a class I heavy chain and β₂-microglobulin that bind peptides supplied by the MHC region-encoded transporters associated with antigen processing (TAP). Peptide binding by class I heterodimers is necessary for their maturation into stable complexes and is dependent on their physical association with TAP. In human mutant 721.220 cells, however, a novel genetic defect causes the failure of class I heterodimers to associate with TAP. This deficiency correlates with lack of expression of a glycoprotein, tapasin (TAP-associated glycoprotein), which has been found in association with class I heterodimers and TAP. Employing a transcomplementation analysis, we obtained evidence co-localizing the genetic defect of mutant 220 cells and the structural or a regulatory gene controlling the expression of tapasin on the short arm of chromosome 6, which includes the MHC. Expression of tapasin and the normal interaction of class I heterodimers with TAP are concomitantly restored, indicating the probable function of tapasin as a physical link between these complexes. In further support of this model, the absence of tapasin in mutant 220 cells correlates with reduced class I heterodimer stability, suggesting that tapasin may stabilize class I heterodimers and thereby enhance their association with TAP. These results further implicate tapasin in a mechanism that promotes peptide binding by class I heterodimers through their interaction with TAP. Received: 20 March 1997 / Revised: 2 June 1997